22s-hydroxycholesta-8, 14-diene derivatives with meiosis regulating activity

ABSTRACT

The invention relates to 22S hydroxycholesta-8, 14-diene derivatives having general formula (I) wherein R 1  is OR, OSO 3 H or —NOR; with R being H, (C 1-6 )alkyl or (C 1-6 )acyl; each of R 2  and R 3  is independently hydrogen or (C 1-6 )alkyl; R 4  is hydrogen, (C 1-6 )alkyl or (C 1-6 )acyl; R 5  is hydrogen; or R 5  designates, together with R 6 , an additional bond between the carbon atoms at which R 5  and R 6  are placed; R 6  is hydrogen, hydroxy or halogen, or R 6  designates, together with R 5 , an additional bond between the carbon atoms at which R 6  and R 5  are placed; each or R 7  and R 8  is independently hydrogen or (C 1-4 )alkyl, optionally substituted with OH, (C 1-4 )alkoxy, or halogen; or a pharmaceutically acceptable salt thereof. The compounds of the invention have meiosis activating activity and can be used for the control of fertility.

This application is the 35 U.S.C. §371 filing of PCT/EP99/04099 filedJun. 10, 1999.

FIELD OF THE INVENTION

The invention relates to 22S-hydroxycholesta-8,14-diene derivatives, topharmaceutical compositions containing the same, as well as to the useof these 22S-hydroxycholesta-8,14-diene derivatives for the preparationof a medicament for the control of fertility.

BACKGROUND OF THE INVENTION

Sexual reproduction involves a cyclic alternation of diploid and haploidstates: diploid cells divide by the process of meiosis to form haploidcells, and the haploid cells fuse in pairs at fertilization to form newdiploid cells. The process of meiosis is characterized by two meioticdivisions, unique to both male and female germ cells. During the processtwo cell divisions, following one round of DNA replication, give rise tofour haploid cells from one single diploid cell. Chromosomal crossoverevents, during which paternal and maternal genetic material isexchanged, occur during the prophase of the first meiotic division. Atthe end of the first meiotic division one member of each chromosomepair, composed of two sister chromatids is distributed to each daughtercell. The second meiotic division segregates each sister chromatide intoa separate haploid cell. Male and female germ cells are subject tosimilar meiotic divisions but differ in the regulation of theseprocesses. In the male meiosis is a continuous process in germ cellsderived from a population of immature germ cells, the stem cellspermatogonia. After sexual maturation of the male, spermatogonia fromthis stem cell population embark on meiosis. The first and secondmeiotic division proceed without interruption and eventually give riseto four mature spermatozoa.

In the female, primary oocytes start the first meiotic division alreadyduring the embryonic stage but they remain arrested in the prophase(dictyate stage) until the female becomes sexually mature. Meiosisresumes at the time of ovulation (egg maturation) after which the firstmeiotic division is completed and the second meiotic division isinitiated. In most vertebrates the second meiotic division is arrestedat the metaphase and only completed after fertilization. At the end ofthe first and of the second meiotic division the cytoplasm dividesasymmetrically to produce two secondary oocytes, each with a haploidnumber of single chromosomes, but greatly differing in size: one is asmall polar body, which eventually degenerates, and the other is a largecell containing all the developmental potential. Finally one mature ovumis produced.

The stage at which the developing oocyte is released from the ovary andis ready for fertilization differs in different species. In bothinvertebrates and vertebrates ovarian accessory cells respond topolypeptides (gonadotropins) produced elsewhere in the body so as tocontrol the maturation of the oocyte and eventually (in most species)ovulation. In humans the primary oocytes of the newborn female arearrested in prophase of meiotic division I and most are surrounded by asingle layer of follicle cells; such an oocyte with its surroundingcells constitute the primordial follicle. A small portion of primordialfollicles sequentially begins to grow to become developing follicles:the follicle cells enlarge and proliferate to form a multilayeredenvelope around the primary oocyte; the oocyte itself enlarges anddevelops the zona pellucida, an extracellular matrix consisting largelyof glycoproteins, and cortical granules, specialized secretory vesiclesjust under the plasma membrane in the outer region, the cortex, of theegg cytoplasm [when the egg is activated by a sperm, these corticalgranules release their contents by exocytosis; the contents of thegranules act to alter the egg coat so as to prevent other sperms fromfusing with the egg].

The developing follicles grow continuously and some of them develop afluid-filled cavity, or antrum, to become antral follicles. Developmentof such follicles is dependent on gonadotropins (mainly folliclestimulating hormone -FSH) secreted by the pituitary gland and onestrogens secreted by the follicle cells themselves. Starting atpuberty, a surge of secretion by the pituitary of another gonadotropin,luteinizing hormone (LH), activates a single antral follicle to completeits development: the enclosed primary oocyte matures to complete themeiotic division I as the stimulated follicle rapidly enlarges andruptures at the surface of the ovary, releasing the secondary oocytewithin. As is the case with most mammals, the secondary oocyte istriggered to undergo division II of meiosis only if it is fertilized bya sperm.

Studies on the mechanisms controlling initiation and regulation of themeiotic process in male and female germ cells suggest a role for cyclicnucleotides in mediating meiotic arrest. Spontaneous maturation ofoocytes can be prevented by compounds that maintain elevated cAMP levels[Eppig, J. and Downs, S. (1984) Biol. Reprod. 30: 1-11]. Purines, likeadenosine or hypoxanthine, are thought to be involved in the cAMPmediated maintenance of meiotic arrest [Eppig, J., Ward-Bailey, P. andColeman, D. (1985) Biol. Reprod. 33: 1041-1049]. The presence of ameiosis regulating substance in a culture system of fetal mouse gonadswas first described by Byskov, A. et al (1976) Dev. Biol. 52: 193-200.It was suggested that the concentrations of a meiosis activatingsubstance (MAS) and a meiosis preventing substance (MPS) regulate themeiotic process in concert [Byskov, A. et al. (1994). In “The physiologyof reproduction”, Eds. Knobil, E. and Neill, J., Raven Press, New York].More recently (3β,5α,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS), isolated from human follicular fluid, and(3β,5α,20R)-4,4-dimethylcholesta-8,24-dien-3-ol, isolated from bulltestes, were identified by Byskov, A. et al [(1995), Nature 374:559-562] as endogenous meiosis activating substances in human andbovine, respectively. These sterols proved to be able to activate theresumption of meiosis in cultured cumulus enclosed and naked mouseoocytes.

Derivatives of the endogenous sterols, having either a saturated or anunsaturated cholestane side chain, have been disclosed in theinternational patent applications WO 96/00235, WO97/00884 and WO98/28323(NOVO NORDISK A/S) as meiosis regulating substances. Meiosis regulatingsubstances are compounds that are agonists or antagonists of a naturallyoccurring meiosis activating substance. Thus, they might be used in thetreatment of infertility or for contraception.

A drawback of the compounds described in the first two patentapplications mentioned above is their restricted membrane penetration,which results in accumulation in cell membranes or fatty tissues,thereby restricting their therapeutic potential as fertility controlagents. This unwanted accumulation is most probably due to thefysico-chemical properties of these compounds, which resemble those ofcholesterol, an important constituent of cell membranes [Norum, K. R. etal (1983) Physiological Rev. 63: 1343; Babiker, A. et al (1998)Biochimica & Biophysica Acta 1392: 333].

BRIEF SUMMARY OF THE INVENTION

We have now found, that certain 22S-hydroxy substituted cholestanederivatives, i.e. the 22S-hydroxycholesta-8,14-diene derivatives offormula I given below, are highly active as meiosis activatingcompounds.

DETAILED DESCRIPTION OF THE INVENTION

wherein

R₁ is OR, OSO₃H or ═NOR; with R being H, (C₁₋₆)alkyl or (C₁₋₆)acyl;

each of R₂ and R₃ is independently hydrogen or (C₁₋₆)alkyl;

R₄ is hydrogen, (C₁₋₆)alkyl or (C₁₋₆)acyl;

R₅ is hydrogen; or R₅ designates, together with R₆, an additional bondbetween the carbon atoms at which R₅ and R₆ are placed;

R₆ is hydrogen, hydroxy or halogen; or R₆ designates, together with R₅,an additional bond between the carbon atoms at which R₆ and R₅ areplaced;

each of R₇ and R₈ is independently hydrogen or (C₁₋₄)alkyl, optionallysubstituted with OH, (C₁₋₄)alkoxy, or halogen;

or a pharmaceutically acceptable salt thereof.

An advantage of 22-hydroxycholestanes is their ability to pass cellmembranes which would circumvent the problems described above, seeStocco, D. M. (1997), Endocrine 6: 99-109.

The above 22S-hydroxycholesta-8,14-diene derivatives having the generalformula I are highly active as meiosis activating substances. In fact,they are active in the CEO assay (see below) wherein even the naturalmeiosis activating substance FF-Mas is not. This does not follow at allfrom the prior art. In this respect it is noted that, incidentally, acertain 22R-hydroxy substituted cholestane derivative(22R-hydroxycholesterol) has been disclosed in WO 98/28323, but is anantagonist.

The invention further provides a pharmaceutical composition comprising a22S-hydroxycholesta-8,14-diene derivative having the general formula I.A further aspect of the invention resides in the use of a22S-hydroxycholesta-8,14-diene derivative having the general formula Ifor the manufacture of a medicament for the control of fertility.

The term (C₁₋₆)alkyl as used in the definition of formula I means abranched or unbranched alkyl group having 1-6 carbon atoms, like hexyl,pentyl, butyl, isobutyl, tertiary butyl, propyl, isopropyl, ethyl andmethyl. Likewise, the term (C₁₋₄)alkyl means an alkyl group having 1-4carbon atoms.

The term (C₁₋₆)acyl means an acyl group derived from a carboxylic acidhaving from 1-6 carbon atoms, like hexanoyl, pentanoyl, pivaloyl,butyryl, propanoyl, acetyl and formyl. Also included within thedefinition of (C₁₋₆)acyl are acyl groups derived from dicarboxylicacids, like hemi-glutaroyl, hemi-succinoyl, and hemi-maloyl. A preferred(C₁₋₄)acyl group is hemi-succinoyl.

The term (C₁₋₄)alkoxy means an alkyloxy having 1-4 carbon atoms, likebutyloxy, propyloxy, isopropyloxy, ethyloxy, and, preferably, methyloxy.

The term halogen means F, Cl, Br or I. Cl and F are preferred, F beingmost preferred.

It is understood that the 22S-hydroxycholesta-8,14-diene derivatives ofthe invention have the natural configurations 5α, 10β, 13β, and 17β. Theconfiguration at position 20 of the 22S-hydroxycholesta-8,14-dienederivatives of the invention can be either R or S. Preferred compoundsare those with the 20S configuration.

Even more preferred compounds according to the invention are the22S-hydroxycholesta-8,14-diene derivatives of formula I wherein R₁ is ORwherein R has the previously given meaning. Among these preferredcompounds those with the 3-OR substituent in the β-configuration areespecially preferred. A specifically preferred agonistic compound of theinvention is the 22S-hydroxycholesta-8,14-diene derivative(3β,5α,20S,22S)-4,4-dimethylcholesta-8,14,24-triene-3,22-diol.

The 22S-hydroxycholesta-8,14-diene derivatives of this invention havethe natural configurations 5α, 10β, 13β, 17β, and possess also one ormore additional chiral carbon atoms. The compounds may therefore beobtained as a pure diastereomer, or as a mixture of diastereomers.Methods for obtaining the pure diastereomers are well known in the art,e.g. crystallization or chromatography.

The meiosis activating activity of the 22S-hydroxycholesta-8,14-dienederivatives of the invention is measured in an in vitro oocyte assay asthe ability to overcome the hypoxanthine maintained meiotic arrest indenuded oocytes (DO) or cumulus enclosed oocytes (CEO).

The compounds can be used to stimulate meiosis in both male and femaleand thus can be used as fertility regulating agents. Fertilityregulation comprises contraception and infertility treatment.

For female contraception a 22S-hydroxycholesta-8,14-diene derivativeaccording to formula I can be used for induction of premature maturationof oocytes which are still inside the ovary, before the naturallyoccurring gonadotropin surge [reduced fertility by inducing prematurematuration of oocytes has been demonstrated in rats by Mattheij, J. etal (1993), Gynecolo Obstet. Invest. 36: 129-135]. On in vivoadministration the compounds of the invention specifically affect germcells and therefore have the advantage of maintenance of endogenoushormonal levels and subsequently maintenance of normal cycle length.Such a contraceptive method will not cause unwanted side-effectssometimes associated with steroidal contraception (e.g. thrombosis,mood, unscheduled bleeding, malignant breast disease).

A further advantage of the 22S-hydroxycholesta-8,14-diene derivatives ofthe invention is their inability to induce maturation in incompetentoocytes (isolated from pre-antral follicles), which indicates that thecompounds will not affect the entire oocyte reserve in the ovaries. Onlyoocytes from antral follicles (competent oocytes) can be induced tomature by the compounds of the invention.

For treatment of female infertility caused by the absence of matureoocytes the compounds of the invention can be administered in vivo totimely stimulate the maturation of competent oocytes.

The compounds of the invention can also be used for suppletion ofculture media for in vitro fertilization procedures in order to improveoocyte quality.

For treatment of male infertility caused by a shortage of the number ofmature spermatozoa the compounds of the invention can be administered invivo to stimulate the maturation of spermatogonia.

For therapeutic use, salts of the compounds of formula I are thosewherein the counterion is pharmaceutically acceptable. However, salts ofthe acids according to formula I [i.e. compounds wherein R₁ is OSO₃H]may also find use, for example, in the preparation or purification of apharmaceutically acceptable compound. All salts, whetherpharmaceutically acceptable or not, are included within the ambit of thepresent invention. Examples of salts of acids according to the inventionare mineral salts such as sodium salt, potassium salt, and salts derivedfrom organic bases like ammonia, imidazole, ethylenediamine,triethylamine and the like.

The compounds of formula I or a pharmaceutically acceptable saltthereof, also referred to herein as the active ingredient, may beadministered enterally or parenterally. The exact dose and regimen ofadministration of the active ingredient, or a pharmaceutical compositionthereof, will necessarily be dependent upon the therapeutic effect to beachieved (contraception or infertility), and will vary with theparticular compound, the route of administration, and the age andcondition of the individual subject to whom the medicament is to beadministered.

In general parenteral administration requires lower dosages than othermethods of administration which are more dependent upon adsorption.However, a dosage for humans preferably contains 0.0001-25 mg per kgbody weight. The desired dose may be presented as one dose or asmultiple subdoses administered at appropriate intervals throughout theday, or, in case of female recipients, as doses to be administered atappropriate daily intervals throughout the menstrual cycle. The dosageas well as the regimen of administration may differ between a female anda male recipient.

In case of in vitro or ex vivo applications, the compounds of theinventions are to be used in the incubation media in a concentration ofapproximately 0.01-5 μg/ml.

The present invention thus also relates to pharmaceutical compositionscomprising a 22S-hydroxycholesta-8,14-diene derivative according toformula I in admixture with pharmaceutically acceptable auxiliaries, andoptionally other therapeutic agents. The auxilliaries must be“acceptable” in the sense of being compatible with the other ingredientsof the composition and not deleterious to the recipients thereof.

Pharmaceutical compositions include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The compositions may be prepared by anymethod well known in the art of pharmacy, for example, using methodssuch as those described in Gennaro et al., Remington's PharmaceuticalSciences (18th ed., Mack Publishing Company, 1990, see especially Part8: Pharmaceutical Preparations and Their Manufacture). Such methodsinclude the step of bringing in association the active ingredient withany auxilliary agent. The auxilliary agent(s), also named accessoryingredients, include those conventional in the art (Gennaro, supra),such as, fillers, binders, diluents, disintegrants, lubricants,colorants, flavoring agents and wetting agents.

Pharmaceutical compositions suitable for oral administration may bepresented as discrete dosage units such as pills, tablets or capsules,or as a powder or granules, or as a solution or suspension. The activeingredient may also be presented as a bolus or paste. The compositionscan further be processed into a suppository or enema for rectaladministration.

For parenteral administration, suitable compositions include aqueous andnon-aqueous sterile injection. The compositions may be presented inunit-dose or multi-dose containers, for example sealed vials andampoules, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of sterile liquid carrier, for example,water prior to use.

Compositions, or formulations, suitable for administration by nasalinhalation include fine dusts or mists which may be generated by meansof metered dose pressurised aerosols, nebulisers or insufflators.

The 22S-hydroxycholesta-8,14-diene derivatives of the invention can alsobe administered in the form of implantable pharmaceutical devices,consisting of a core of active material, encased by a releaserate-regulating membrane. Such implants are to be applied subcutaneouslyor locally, and will release the active ingredient at an approximatelyconstant rate over relatively large periods of time, for instance fromweeks to years. Methods for the preparation of implantablepharmaceutical devices as such are known in the art, for example asdescribed in European Patent 0,303,306.

The compounds of the invention may be produced by various methods knownin the art of organic chemistry in general, and especially in the art ofthe chemistry of steroids (see, for example: Fried, J. and Edwards, J.A., “Organic Reactions in Steroid Chemistry”, Volumes I and II , VanNostrand Reinhold Company, New York, 1972). A convenient startingmaterial for the preparation of compounds of formula I is a compound ofgeneral formula II ,

wherein R₂ and R₃ are independently hydrogen or (C₁₋₆)alkyl, R₉ is ahydroxy-protecting group such as an acyl group, like an acetyl group, abenzoyl group or a pivaloyl group, or an alkoxyalkyl group, like anethoxyethyl group or a tetrahydropyranyl (THP) group, whose preparationis described in WO-09852965 and WO-09855498. Suitable protective groupsare known in the art [for example from Greene, T. W. and Wuts, P. G. M.:Protective Groups in Organic Synthesis, Second Edition, Wiley, N.Y.,1991.

Starting from this key intermediate, the side-chain is constructed,using methods known in the art [see: Redpath, J. et al, Chem. Soc. Rev.12, 75 (1983); Zhu, G.-D. et al, Chem. Rev. 95, 1877 (1995); Apfel, M.A., J. Org. Chem. 44, 643 (1979); Kircher, H. W. et al, J. Org. Chem.52, 2586 (1987); Takeshita, T. et al, Chem. Pharm. Bull. 24, 1928(1976); Dasgupta, S. K. et al, J. Org. Chem. 39, 1658 (1974); Dolle, R.E. et al, J. Am. Chem. Soc. 111, 278 (1989); Poyser, J. P. et al, J.Chem. Soc., Perkin Trans. I, 2061 (1974)].

For instance, compounds of formula I (R₁═OH, R₄═H, R₅═H) can be preparedby reaction of compounds of formula II with a suitably substitutedalkylmetallic reagent of formula III,

wherein M is Li, MgX, ZnX, or CeX (X═Cl,Br,I), R₇ and R₈ have thepreviously given meaning, any hydroxy group present in R₇ or R₈ beingsuitably protected, and R₁₀ is H, protected OH, or halogen, which oftenresults in the predominant formation of the 22S-hydroxycholestanederivative [see e.g. Poyser, J. P. et al, J. Chem. Soc., Perkin Trans.I, 2061 (1974)]. The 22S-hydroxy- and 22R-hydroxy derivatives can beseparated or, if necessary, the 22R-hydroxy isomer is epimerized to the22S-hydroxy compound. Epimerization at C-22 can be accomplished e.g. bymeans of a Mitsunobu reaction [see Hughes, D. L., Organic Reactions 42,335 (1992)], or by treatment with methanesulfonyl chloride orp-toluenesulfonyl chloride followed by reaction with an oxygennucleophile [e.g. potassium superoxide, see Corey, E. J. et al,Tetrahedron Lett. 3183 (1975) and Larock, R. C., “Comprehensive OrganicTransformations”, VCH Publishers, Inc.,1989, p. 479]. In both cases,removal of any remaining protective groups then results in compounds offormula I (R₁═OH; R₄,R₅═H).

Compounds of formula I (R₁═OH, R₄═H, R₅ and R₆ together form anadditional bond, i.e. a Δ²⁴ double bond) can be obtained from aldehydeII as follows: the latter is reacted with the anion of acetonitrile[MCH₂C≡N, M═Li, Na, K, MgX, ZnX; see: Arseniyadis, S. et al, Org. React.31, 1 (1984)] to give a diastereomeric pair of 22R and22S-hydroxycholane-24-nitrile derivatives. After isolation of the22S-hydroxy epimer the 22-hydroxy group is protected as a silyl ether oras an alkoxyalkyl ether. If necessary, the 3-hydroxy group isreprotected in the same way, or with an orthogonal protective group. Thecyano group is reduced to the corresponding carboxaldehyde group bytreatment with a reducing agent such as diisobutylaluminium hydride orother reducing agents capable of converting a carbonitrile group into acarboxaldehyde group. Wittig reaction with a suitably substituted Wittigreagent and removal of protective groups then results in thecholest-24-enes of formula I (R₁═OH, R₄═H, R₅ and R₆ together form a Δ²⁴double bond).

For methods used for the Wittig olefination reaction, see Maercker, A.,Org. React. 14, 270 (1965). Alternatively, Peterson reactions can beused, see Ager, D. J. Org. React. 38, 1 (1990).

The Wittig reaction can also be performed in the opposite direction. Inthat case, the cholan-24-al derivative mentioned above is reduced to thecorresponding cholan-24-ol derivative with the use of reducing agentslike for example lithium aluminium hydride, sodium borohydride, or otherhydride reducing agents known in the art. The 24-hydroxy group isconverted to a leaving group, e.g. Br, I, mesyloxy, or tosyloxy.Reaction with an appropriate phosphine (e.g. triphenyl phosphine),Wittig reaction with a suitably substituted ketone, and finally, removalof protective groups then results in the formation of compounds offormula I (R₁═OH, R₄═H, R₅ and R₆ together form a Δ²⁴ double bond).

Construction of Δ²⁴-cholestanes I from aldehydes II can also beaccomplished by an analogous reaction sequence which makes use of anionsof acetic acid or anions of acetic acid esters [see: Petragnani, N. etal, Synthesis, 521 (1982)]. Techniques for homologation are known in theart, see for example Mathieu, J. et al: Formation of C—C Bonds, Vol.I-III, Georg Thieme Publishers, Stuttgart, 1973.

Selective deprotection of 3-OH and 22-OH enables independent conversion,using methods known in the art, of 3-OH into 3-OR, OSO₃H or ═NOR (R aspreviously defined), and of 22-OH into 22-OR₄ (R₄ as previouslydefined), respectively.

The invention is further illustrated by the following examples.

EXAMPLE 1 (3β,5α,20S ,22S)-4,4-Dimethylcholesta-8,14-diene-3,22-diol

i)—A solution of 3-methylbutylmagnesium iodide (12 ml), prepared from1-iodo-3-methylbutane (2.36 ml) and magnesium (0.48 g), activated with1,2-dibromoethane (0.040 ml), in diethyl ether (10 ml and 10 ml,respectively) at reflux temperature, was added dropwise to(3β,5α,20S)-3-(benzoyloxy)-4,4-dimethylpregna-8,14-diene-20-carboxaldehyde(WO-09852965; 1.55 g) in THF (20 ml). The reaction mixture was stirredat room temperature for 45 min. and then poured into a saturated aqueoussolution of ammonium chloride. The product was extracted into ethylacetate; the combined organic phases were washed with a saturatedaqueous solution of ammonium chloride and with brine, dried overmagnesium sulfate and concentrated under reduced pressure. Columnchromatography afforded(3β,5α,20S,22S)-4,4-dimethylcholesta-8,14-diene-3,22-diol 3-benzoate(1.1 g).

ii)—A solution of the product obtained under i (0.35 g) in drytetrahydrofuran (5 ml) was added dropwise to an ice-cooled suspension oflithium aluminium hydride (0.075 g) in dry tetrahydrofuran (5 ml). Themixture was stirred at room temperature for 30 min. The reaction mixturewas cooled to 0° C., and then quenched with a saturated aqueous solutionof sodium sulfate. Then it was filtered over dicalite and the filtrateconcentrated under reduced pressure to give, after columnchromatography,(3β,5α,20S,22S)-4,4-dimethylcholesta-8,14-diene-3,22-diol (0.18 g), m.p.142-143° C.

EXAMPLE 2 (3β,5α,20S,22S )-4,4-Dimethylcholesta-8,14,24-triene-3,22-diol

i)—Tetrapropylammonium perruthenate (0.180 g) was added to a solution of(3β,5α,20S)-4,4,20-trimethylpregna-8,14-diene-3,21-diol 3-benzoate(WO09852965; 4.62 g) and 4-methylmorpholine N-oxide (3.50 g) in acetone(80 ml). After 30 min. stirring at room temperature the reaction mixturewas filtered over dicalite and silica. The filtrate was concentratedunder reduced pressure, to give(3β,5α,20S)-3-(benzoyloxy)-4,4-dimethylpregna-8,14-diene-20-carboxaldehyde(4.28 g) which was used in the following step without furtherpurification.

ii)—A solution of diisopropylamine (8.40 ml) in dry THF (100 ml) wascooled to −30° C. n-BuLi (1.6 M solution in hexanes, 37.6 ml) was addeddropwise while the temperature was allowed to raise to −5° C. Stirringwas continued for 10 min. whereafter the reaction mixture was cooled to−78° C. Acetonitrile (3.14 ml) was added dropwise in 5 min. and stirringwas continued for another 30 min. A solution of the aldehyde obtained inthe previous step (4.28 g) in THF (60 ml) was added dropwise and themixture was stirred at −78° C. for 2 h. Then it was poured into asaturated aqueous solution of ammonium chloride and the product wasextracted into ethyl acetate. The combined organic phases were washedwith brine, dried over sodium sulfate and concentrated under reducedpressure. Column chromatography afforded(3β,5α,20S,22R)-3,22-dihydroxy-4,4-dimethylchola-8,14-diene-24-nitrile(1.23 g) and(3β,5α,20S,22S)-3,22-dihydroxy-4,4-dimethylchola-8,14-diene-24-nitrile(0.85 g).

iii)—Pyridinium p-toluenesulfonate (0.20 g) was added to a solution of(3β,5α,20S,22S)-3,22-dihydroxy-4,4-dimethylchola-8,14-diene-24-nitrile(0.70 g), in dichloromethane (8 ml) and ethyl vinyl ether (4 ml). Afterstirring of the reaction mixture for 1 h at room temperature the mixturewas poured into a saturated aqueous solution of sodiumhydrogencarbonate. The product was extracted into diethyl ether; thecombined organic phases were washed with brine, dried over sodiumsulfate, and concentrated under reduced pressure to give(3β,5α,20S,22S)-3,22-bis[(1-ethoxyethyl)oxy]-4,4-dimethylchola-8,14-diene-24-nitrile(1.0 g), which was used in the following step without furtherpurification.

iv)—Diisobutylaluminium hydride (20% solution in toluene, 8 ml) wascooled to −78° C. A solution of the compound obtained in the previousstep (1.0 g) in dry THF (10 ml) was added dropwise and stirring wascontinued for 15 min. at −78° C. and then for 1.5 h at 0° C. Thereaction was quenched with an aqueous solution of acetic acid (20%, 0°C.). The reaction mixture was poured into a saturated aqueous solutionof sodium hydrogencarbonate and the product was extracted into diethylether. The combined organic phases were washed with brine, dried oversodium sulfate, and concentrated under reduced pressure to give(3β,5α,20S,22S)-3,22-bis[(1-ethoxyethyl)oxy]-4,4-dimethylchola-8,14-dien-24-al(0.96 g), which was used in the following step without furtherpurification.

v)—A suspension of i-propyltriphenylphosphonium iodide (4.57 g) in dryTHF (20 ml) was cooled to −30° C. n-BuLi (1.6 M solution in hexanes,6.50 ml) was added dropwise whereafter the temperature was allowed toraise to 0° C. in 30 min. Stirring was continued for 30 min. at 0° C.and then for another 30 min. at room temperature. After cooling to −30°C., a solution of the aldehyde obtained in the previous step (0.96 g) inTHF (15 ml) was added and the mixture was stirred for 1 h while thetemperature was allowed to raise to room temperature. Then it was pouredinto a saturated aqueous solution of ammonium chloride and the productwas extracted into diethyl ether. The combined organic phases werewashed with brine, dried over sodium sulfate and concentrated underreduced pressure, to give a mixture of(3β,5α,20S,22S)-3,22-bis[(1-ethoxyethyl)oxy]-4,4-dimethylcholesta-8,14,24-triene,phosphonium salt and triphenylphosphine oxide (1.86 g), which was usedin the following step without further purification.

vi)—A mixture of silica (12 g), a saturated aqueous solution of oxalicacid (2 ml), and dichloromethane (20 ml) was stirred at room temperaturefor 10 min. A solution of the product obtained in the previous step(1.86 g) in dichloromethane (10 ml) was added and stirring was continuedfor 3 h. The reaction mixture was filtered and the filtrate wasconcentrated under reduced pressure. Column chromatography of the crudeproduct afforded(3β,5α,20S,22S)-4,4-dimethylcholesta-8,14,24-triene-3,22-diol (0.25 g),m.p. 106-108° C.

EXAMPLE 3 The Oocyte Assay

General:

Oocytes arrested in meiosis contain diffused chromosomes which aresurrounded by an intact nuclear envelope known as the germinal vesicle(GV). Upon reinitiation of meiosis by the midcycle gonadotropin surge,the chromosomes recondense and the GV breaks down (GVBD). In vivo, theoocyte is exposed to hypoxanthine (HX), which maintains the oocytearrested in the meiotic prophase. This meiotic arrest can be mimicked invitro by addition of hypoxanthine to the culture medium. The agonisticactivity of the meiosis activating substances is measured as the abilityto overcome the hypoxanthine maintained meiotic arrest in denudedoocytes (DO) or cumulus enclosed oocytes (CEO), i.e. as the ability toinduce meiotic resumption in vitro.

Isolation of Cumulus Enclosed Oocytes:

Ovaries are obtained from immature female mice (B6D2-F1, strainC57BL×DBA). At the age of 19, 20 or 21 days the mice are injectedsubcutaneously with a single dose of 20 IU follicle stimulating hormone(Humegon, Organon, The Netherlands) in saline.

Forty-eight hours after follicle stimulating hormone injection mice arekilled by cervical dislocation. The ovaries are removed, freed ofextraneous tissue and placed in a multidish containing 0.5 mlpreparation medium at 37° C. L-15 Leibovitz medium (Gibco, pH 7.3±0.1)supplemented with bovine serum albumin (3 mg.ml⁻¹), L-glutamine (0.23mM), sodium pyruvate (2 mM) and hypoxanthine (4 mM) is used aspreparation medium. The antral follicles of the ovaries are puncturedunder a dissecting microscope using two 27-gauge needles attached to two1 ml syringes. Cumulus enclosed oocytes (CEO) of uniform size areselected with a mouth-controlled pipette and rinsed in 0,5 ml freshpreparation medium. About 20 CEO are obtained from one ovary.

Isolation of Denuded Oocytes:

Oocytes freed from cumulus cells, i.e. denuded oocytes (DO), areobtained by gently flushing CEO through a fine-bore mouth-controlledpipette. DO were collected in fresh MEM alpha medium, containing bovineserum albumin (3 mg.ml³¹ ¹), L-glutamine (0.23 mM), sodium pyruvate (2mM) and hypoxanthine (4 mM), and washed twice before transfer to thetest medium.

Experimental Design:

The oocyte assay is performed in 3 blocks, each block represents theovaries of one mouse (randomized block design). At t=0 DO or CEO of thefirst ovary of the first mouse, are spread over well 1 and 3 and oocytesof the second ovary over well 2 and 4 of a 4-well multidish containing0.5 ml of culture medium to which a 22S-hydroxycholesta-8,14-dienederivative of the invention is added [first block]. Culture medium wasused as control. The same procedure is performed for the second andthird mouse [block 2 and 3]. The culture medium used is MEM alpha medium(Gibco, pH 7.3±0.1) saturated with CO₂ and supplemented with bovineserum albumin (3 mg.ml⁻¹), L-glutamine (0.23 mM), sodium pyruvate (2 mM)and hypoxanthine (4 mM). In total, each control or test compound istested on 30 oocytes (10 oocytes per block). At t=0 the number ofoocytes with intact germinal vesicles (GV) or germinal vesiclebreak-down (GVBD) is counted under an inverted microscope withdifferential interference contrast equipment. Only oocytes with anintact GV are used in the experiment. Oocytes are cultured 22 hours at37° C. in 100% humidified atmosphere with 5% CO₂ in air. At the end ofthe culture period the number of oocytes with GV or GVBD per group iscounted. For statistical analysis the percentage germinal vesiclebreakdown is calculated for each group in one block. These percentagesare subjected to arcsin transformation, and differences between controland test compounds are analyzed by an ANOVA test for a randomized blockdesign. Results are presented in Table I.

TABLE I Percentage germinal vesicle breakdown (GVBD) in oocytesfollowing culturing in the presence of test compounds in agonistictesting. % GVBD CEO DO assay assay exp. exp. Compound¹ (control)(control) (3β,5α,20S,22S)4,4-dimethylcholesta-8,14- 85(0)  6(3)diene-3,22-diol (Example 1)(3β,5α,20S,22S)-4,4-dimethylcholesta-8,14,24- 97(2) 73(2)triene-3,22-diol (Example 2)(3β,5α,20R)4,4-dimethylcholesta-8,14,24-trien- 84(7)  0(0) 3-ol(FF-Mas)² ¹Each compound was tested at a concentration of 5 μM. ²Testedat a concentration of 10 μM.

What is claimed is:
 1. A meiosis activating compound having the generalformula I:

wherein R₁ is OR, OSO₃H or ═NOR; with R being H, (C₁₋₆)alkyl or(C₁₋₆)acyl; each R₂ and R₃ is independently hydrogen or (C₁₋₆)alkyl; R₄is hydrogen, (C₁₋₆)alkyl or (C₁₋₆)acyl; R₅ is hydrogen; or R₅designates, together with R₆, an additional bond between carbon atoms atwhich R₅ and R₆ are placed; R₆ is hydrogen, hydroxy or halogen; or R₆designates, together with R₅, an additional bond between carbon atoms atwhich R₆ and R₅ are placed; each R₇ and R₈ is independently hydrogen or(C₁₋₄)alkyl; or a pharmaceutically acceptable salt thereof.
 2. Themeiosis activating compound according to claim 1, wherein C-20 has the20S configuration.
 3. The meiosis activating compound of claim 2,wherein R₁ is OR, wherein R is H or (C₁₋₆)acyl, and the configuration ofthe 3-OR substituent is the β-configuration.
 4. A22S-hydroxycholesta-8,14-diene derivative selected from the groupconsisting of (3β,5α,20S,22S)-4,4-dimethylcholesta-8,14-diene-3,22-dioland (3β,5α,20S,22S)-4,4-dimethylcholesta-8,14,24-triene-3,22-diol.
 5. Amethod for meiosis activation, comprising: administering an effectiveamount of the meiosis activating compound according to claim
 1. 6. Apharmaceutical composition, comprising: a meiosis activating compoundaccording to claim 1 in admixture with at least one pharmaceuticallyacceptable auxiliary.
 7. The meiosis activating compound of claim 1,wherein each of R₇ and R₈ is independently (C₁₋₄)alkyl substituted withOH, (C₁₋₄)alkoxy, or halogen.
 8. The meiosis activating compoundaccording to claim 1, wherein R is selected from the group consisting ofhydrogen, a hexyl group, a pentyl group, a butyl group, an isobutylgroup, a tertiary butyl group, a propyl group, an isobutyl group, atertiary butyl group, a propyl group, an isopropyl group, an ethylgroup, a methyl group, a hexanoyl group, a pentaoyl group, a pivaloylgroup, a butyrl group, a propanoyl group, an acetyl group, a formylgroup, a hemi-glutaroyl group, hemi succinoyl group, and a hemi-maloylgroup.
 9. The meiosis activating compound according claim 1, whereineach of R₂ and R₃ is selected from the group consisting of hydrogen, ahexyl group, a pentyl group, a butyl group, an isobutyl group, atertiary butyl group, a propyl group, an isopropyl group, an ethylgroup, and a methyl group.
 10. The meiosis activating compound accordingto claim 1, wherein R₄ is selected from the group consisting ofhydrogen, a hexyl group, a pentyl group, a butyl group, an isobutylgroup, a tertiary butyl group, a propyl group, an isopropyl group, anethyl group, a methyl group, a hexanoyl group, a pentaoyl group, apivaloyl group, a butyrl group, a propanoyl group, an acetyl group, aformyl group, a hemi-glutaroyl group, hemi succinoyl group, and ahemi-maloyl group.
 11. The meiosis activating compound according toclaim 7, wherein each of R₇ and R₈ is independently selected from thegroup consisting of hydrogen, a butyl group, an isobutyl group, atertiary butyl group, a propyl group, an isopropyl group, an ethylgroup, and a methyl group.